Each of the references cited herein is incorporated by reference in its entirety. A complete listing of the citations is set forth at the end of the specification.
Adult soft tissue sarcomas constitute a family of relatively uncommon tumors. They account for approximately 1% of the cancer diagnoses each year in the United States (1). Sarcomas consist of a heterogeneous group of histologically distinct malignancies that arise from mesenchymal tissues. Liposarcomas are, for example, malignant tumors derived from primitive or embryonal lipoblastic cells and are histologically distinct from Kaposi's sarcoma, an indolent vascular tumor.
Liposarcomas are the second most common adult soft tissue sarcoma, accounting for approximately 20% of all sarcomas. They range from low-grade, well-differentiated, and myxoid liposarcomas to high-grade, round cell, and pleomorphic liposarcomas (2). Well-differentiated liposarcomas can exhibit aggressive local invasion but rarely metastasize until late in their course when they progress to high-grade undifferentiated sarcomas of other histologic origin (i.e., leiomyosarcoma, rhabdomyosarcoma, or even osteosarcoma). Round cell and pleomorphic liposarcomas have a high potential for distant metastasis and survival rates are poor, with 5-year survival of 20-30% in patients with these tumors (3). Liposarcomas may also be a feature of other conditions including but not limited to Type 1 Carney Complex (30, 31) or multiple lipomatosis (31). Most clinical trials of chemotherapy for advanced soft tissue sarcomas do not differentiate liposarcomas from other soft tissue sarcomas. Single-agent doxorubicin chemotherapy for advanced soft tissue sarcomas yields a modest response rate of about 25% (3). Doxorubicin in combination with ifosfamide typically induces responses of 20-40% in less-differentiated tumors. Unfortunately, in a large U.S. cooperative group study of the combination of doxorubicin, ifosfamide, and dacarbazine for advanced soft tissue sarcomas, the response rate of 32% was only modestly better than the response rate for doxorubicin and dacarbazine alone (17%) (29). The median time to progression was modestly improved from 4 to 6 months. Severe and life-threatening toxicities were more common, however (55% vs. 88%) (29).
Surgery, alone or in combination with chemotherapy and/or radiotherapy, remains the primary treatment modality for liposarcomas. Metastatic liposarcoma is associated with an extremely poor prognosis, with average 5-year survivals ranging from 70% to as low as 25% depending on the type of tumor. Conventional chemotherapy for recurrent liposarcomas or for metastatic liposarcoma leads to a complete response in only about 10% of patients, and thus, is largely palliative (4, 5). Novel, targeted, and less toxic therapies are urgently needed.
Induction of terminal differentiation represents one approach in the search for novel targeted therapeutic agents for certain malignancies. For example, the nuclear retinoic acid receptor α (RARα), which plays an important role in the differentiation and malignant transformation of cells of the myelocytic lineage, is a target for intervention in acute promyelocytic leukemia (APL) (6). Differentiation therapy with all-trans retinoic acid has become the standard of care for this disease (7).
Similar to RARα in APL, the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) and the retinoid X receptor α (RXRα) form a heterodimeric complex that functions as a central regulator of adipocyte differentiation. Primary human liposarcoma cells express high levels of PPARγ and can be induced to undergo terminal differentiation by treatment with PPARγ ligands such as the antidiabetic medication, pioglitazone (a 2,4-thiazolidinedione) (8).
A clinical trial of a different thiazolidinedione, troglitazone, was conducted for treatment of patients with intermediate to high-grade liposarcoma (9). Biopsies of tumors treated with troglitazone demonstrated evidence of differentiation and reduction of cellular proliferation. Although the long-term effect of thiazolidinediones on liposarcomas requires further study, the toxicity of therapy was negligible, and one patient exhibited temporary disease stabilization. These studies demonstrate that targeted therapy for solid tumors such as liposarcomas is viable.
The development of highly active antiretroviral therapy (HAART) has significantly improved the outcome of individuals infected with human immunodeficiency virus (HIV) type-1 infection (10). An integral component of HAART is the HIV protease inhibitor. The HIV protease enzyme targets the amino acid sequences in the gag and gag-pol polyproteins, which must be cleaved before nascent viral particles (virions) can mature (11). Proviral DNA lacking functional protease produces immature, noninfectious viral particles (12). HIV protease inhibitors prevent cleavage of gag and gag-pol protein precursors in acutely and chronically infected cells, arresting maturation and thereby blocking the infectivity of nascent virions (11). Four HIV protease inhibitors, indinavir, nelfinavir, ritonavir, and saquinavir, are structurally related, but differ based upon the amino acid sequences recognized and cleaved in HIV proteins (11).
An unanticipated consequence of HAART has been the development of a distinct clinical syndrome consisting of peripheral lipoatrophy and central fat accumulation associated with insulin resistance and hyperlipidemia, which is directly linked to the use of the HIV protease inhibitor (13). This clinical syndrome is labeled “HIV protease-induced lipodystrophy syndrome” (14). The pathophysiology of HIV protease-induced lipodystrophy syndrome is currently under intense investigation and debate.
One mechanism proposed to explain the pathophysiology of the syndrome is the inhibition of adipocyte differentiation via inhibition of PPARγ (15). Another theory is the alteration in the level of sterol regulatory element binding protein 1 (SREBP-1) (16-18). SREBP-1 is a member of the basic helix-loop-helix-leucine zipper transcription factor family (19). It promotes lipogenic gene expression (19) and stimulates production of an unidentified PPARγ ligand (20). Thus, SREBP-1 and PPARγ cooperatively promote adipogenesis and subsequent maintenance of the adipocyte phenotype. Consistent with this hypothesis, HIV protease inhibitors have been shown to induce lipolysis by reducing levels of SREBP-1 (21). Further, this property of HIV protease inhibitors seems to target differentiated adipocytes selectively. It has been demonstrated that incubation with ritonavir, saquinavir and nelfinavir for 48 hours induced apoptosis in differentiated adipocytes, but not in pre-adipocytes (21).
Proteases are important components in the replication of a number of viruses and viral protease inhibitors represent a growing class of anti-viral therapeutics. A number of HIV protease inhibitors are currently in the clinic, and protease inhibitors for additional viruses are under investigation.
Thus, there is a serious need for improvement of treatment methods for liposarcoma patients. If lipodystrophy were observed with the clinical use of a protease inhibitor, then the findings would indicate that such a protease inhibitor may also be useful for the treatment of liposarcoma. The present invention addresses this need with novel methods of use of inhibitors of viral proteases.